Histoplasmosis in Malaya

Brit. 3. Dis. Chest (1964) 58, 49

HISTOPLASMOSIS IN MALAYA B Y J. T. P O N N A M P A L A M From the Department of Bacteriology,Institutefor Medical Research, Kuala Lumpur, Malaya

UP to now no data have been available for estimating the prevalence and distribution of histoplasmin sensitivity in Malaya. The possibility of histoplasmosis being present as a disease entity here was suggested by the isolation of Histoplasma capsulatumfrom the soil of a cave infested with bats, located in a limestone cliff in Central Malaya (J.T.P., 1963). I n order to obtain this information a survey was carried out on adult patients and children admitted to Lady Templer Hospital, Kuala L u m p u r . This hospital provides medical and surgical treatment for patients referred from chest clinics all over Malaya from a population of about 7 millions. MATERIALS AND METHODS

T h e study was based on inpatients at the hospital. All received both histoplasmin and tuberculin. Stock histoplasmin, H K C - 5 2 : IOOO, was supplied by the Communicable Disease Centre, United States Public Health Service. For use o. I ml. antigen is added to 49"9 ml. diluent (sterile normal saline) giving 5 ° II11. of H K C - 5 2:lOOO. This is the recommended dilution for h u m a n use. Diluted histoplasmin stored at 4 ° C. may be used for one month. Stock histoplasmin at 4 ° C. lasts indefinitcly. T h e tuberculin used was diluted at the Institute for Medical Research from stock solution of tuberculin, R T 23 supplied by the Statcns Seruminstitut, Copenhagen. Diluted tuberculin contains I T.U. in o" I ml. o.I ml. each of tuberculin and histoplasmin were given intradermally, applying the histoplasmin in the right upper forearm and the tuberculin in the same site on the left forearm. Tests were read after 48 hours. All reactions with an induration of 5 ram. or more were considered positive. Different syringes were used for the Mantoux and histoplasmin skin tests. Needles were also kept separatcly and sterilized separately and different trays were used for the histoplasmin and tuberculin syringes at the time of inoculation. This was done to avoid cross-reactions and false positives. T h e following information was obtained for all patients irrespective of the results of the skin tests: (I) Clinical history. (2) Chest X-ray with repeat examination at monthly intervals. (3) Examination of sputum for Mycobacteriumtuberculosis. (4) Serological studies (5 ° per cent. end-point complement fixation tests) using histoplasmin and whole yeast antigen. (ReceivedJbr publication December2, 1963.) VOL. LVIII. 2

I

PONNAMPALAM

5 °

(5) Examination of sputum for Histoplasma ¢apsulatum from patients with a positive complement fixation test provided they were still in hospital and had not returned to their homes in distant parts of the country where it was impossible to do any follow-up work. RESULTS

Two hundred and twenty-seven adults and children were studied, females 57, males 167, comprising the three major races of the country, viz. Chinese, Malays, Indians. Of this number 36 were Indians, 51 Malays, and 15o Chinese. The ages varied from 5-6o years and were divided into 6 groups, viz. Group I, age 5-IO years; Group II, age 11-3o years; Group III, age 31-3o years; Group IV, age 31-4 ° years; Group V, age 41-5 ° years, and Group VI, age 51-6o years. The largest numbers of patients were in Groups I I I and IV. See Tables I, II, and III. TABLE I.~X-RAY FINDINGS: M A L A Y S

Age in years

Numbers tested

Histoplasmin skin test positive

Complementfixation positive

Tubereulin positive

Sputum positive for Mycobacterium tuberculosis

Calcification

InfiItration

Caviration

10"-`90

6

5

3

o

3

4

21-3o

x5

H

7

x

9

7

3I-4o

x8

3

2

x4

xo

4

I`9

Io

¢x-5o

IO

I

`9

xo

6

o

6

5

5i-6o

.9

0

l

I

2

O

2

I

5I

6

I0

4x

28

5

32

27

Sputum positive for M. tuberculosis

Calebcation

Infil. tration

Caviration

0

~OTAL:

TABL~ II.--X-RAY FmmNos: I N D I A N S

Age in years

Number tested

Histoplasmin skin test positive

Complement. Tuber. fixation culin positive positive

5-xo

`9

0

0

2

0

0

io-`9o

5

0

0

4

o

O

2x- 3 °

4

I

0

4

2

0

4

3

3I-4o

7

0

0

6

4

0

6

x

¢x-5o

4

0

I

4

3

0

3

3

4

0

0

4

o

0

3

x

26

I

I

`94

9

0

x8

io

5t-6o ~OTAL:

1

x

HISTOPLASMOSIS IN MALAYA

51

' TABL~ I I I . - - X - ~ Y

Fmm~Gs: CHINESE

Number tested

Histoplasmin skin test positive

Complementfixation positive

Tuberculin positive

@utum positive for M , tuberculosis

Calcifi. cation

Infiltration

Cavitation

xo-2o

23

o

4

20

15

3

~i

9

21-3o

46

3

5

36

o7

6

4°

19 ~2

Age in years

3I-4o

4I

6

9

31

3°

3

33

4I-5o

22

4

5

~o

x3

2

i8

5i-6o

i8

4

3

x4

9

3

x3

7

15 °

x7

26

~,,I

94

17

z25

7I

TOTAL :

All patients had had respiratory symptoms for varying periods, and had received treatment in hospitals and clinics in outlying districts or from general practitioners before being referred to Lady Templer Hospital. At the time of admission none had symptoms which suggested infection with Histoplasma capsulatum. One had co-existent diabetes. None of these patients were investigated for histoplasmosis until the survey was started. Radiological examination was carried out at monthly intervals in the early stages after admission, in some patients oftener as individual cases required. The results of the radiological examinations in this survey were from films taken on the first occasion after admission to hospital. The findings were described as calcification, infiltration, and cavitation. Tables I, II, and I I I show the X-ray findings in all three major races against the different age groups. All patients had more than one specimen of sputum examined in outlying chest clinics for Mycobacterium tuberculosisby direct examination and all patients had sputum examination for M. tuberculosisat monthly intervals after admission to Lady Templet Hospital, in addition to examination of sputum at the time of admission, by direct examination, examination of concentrated smears and culture. Complement fixation tests were carried out by the Communicable Disease Centre on the sera of all patients examined, irrespective of the results of the histoplasmin skin test. Twenty-five anticomplementary sera were not included in the series; they were negative for histoplasmosis when tested by the precipitin reaction. Blood for serological studies was collected at the time the skin tests were read. Examination of the sputum for Histoplasma capsulatum was delayed until the results of the complement-fixation tests were known. This serological examination was regarded as a useful screening test since patients with a positive result were considered to be those most likely to reveal H. capsulatum in cultures of sputum. Sputum cultures were therefore limited to such patients. Six earlymorning specimens of sputum were collected from each patient on alternate days over a period of 2 weeks, leaving out the week-end. These were collected

52

PONNAMPALAM

after the p a t i e n t h a d b r u s h e d his teeth, a n d cleansed the m o u t h with clean water. N o antiseptic gargles w e r e used. T h e s p u t u m was b r o u g h t to the laborat o r y at the Institute a n d 2,500 units o f c h l o r a m p h e n i c o l were a d d e d to each ml. o f sputum, I ml. o f w h i c h was i n o c u l a t e d i n t r a p e r i t o n e a l l y i n t o e a c h o f 5 mice w h i c h were p r o t e c t e d with daily injections o f 3,500 units o f c h l o r a m p h e n i c o l for a week. Antibiotic t h e r a p y was necessary to p r o t e c t the animals f r o m overTABLE I V

Reg~te~d No.

4~o4 471o 4336 433° 4553 47o6

Da~

2o June I3 September 24 October IO December I8 January lO December 18 January Io December 18 January 18 March 1o December 18 January lO December 18 January

C.F. (Histoplasmin)

C.F. ( Teast)

8

64

1962 1962 1962 1962 1963 1962 1963 I962 1963 1963 1962 1963 1962 1963

0 O O 0 O O 0 O O 0 O O O

O

32

8

0

8 0

16 I6 0

8 16 8 O

T A B L E V . - - P A T I E N T S IN WHOM THE TUBERCULIN TEST AND SPUTUM FOR M . W ~ I ~ POSITIVE.

Histoplasmin Skin test

Positive Positive Negative Negative

.... .... .... ....

C.F.

Number

Positive Negative Negative Positive

8 83 26

Tuberculosis

I

118

T A B L E V I . - - P A T I E N T S IN WHOM THE TUBERCULIN T E S T WAS NEGATIVE BUT THE SPUTUM WAS

PosrrxvE FOR M. Tuberculosis Histoplasmin Skin Text

Positive Positive Negative Negative

.... .... .... ....

G.F.

Positive Negative Negative Positive

Number O 2

15 2

I9 I n this series 4 o f the 19 patients show evidence o f infection or h a d been exposed to infection at some time to Histoplasma capsulatum.

53

tnSTOPLASMOSlS IN M A L A Y A T A B L E V I I . - - P A T I E ~ S IN W H O M

T H E T U B E R C U L I N TEST W A S POSITIVE BUT T H E S P U T U M NEGATIVI~ FOR M. Tuberculosis

Histoplasmin Skin Text

Positive Positive Negative Negative

.... .... .... ....

C.F.

Number

Positive Negative Negative Positive

3

i

56 x3 73

In this series 4 had a positive skin test and 14 had positive complement-fixation (C.F.) tests. One patient with a positive histoplasmin skin test had a positive C.F. test as well. Three patients with a positive skin test had a negative C.F. test. Thirteen patients with a positive complement-fixation test had negative histoplasmin skin tests. TABLE V I I I . - - P A T I E N T S WITH A N E G A T I ~ TUBERCULIN TEST WHOSE SPUTUM WAS ALSO NEGA-

TIVE FOR M. Tuberculosis Histoplasmin Skin Test Positive Positive Negative Negative

. . . .

. . . .

. . . .

. . . .

C.F,

Number

Positive Negative Negative Positive

17 I n this series 8 patients show evidence of exposure to infection with Histoplasma

capsulatum. whelming infection and death due to the bacteria present in the sputum, and at the same time to allow growth of the fungus in the tissues of the animal. The mice were sacrificed at weekly intervals at the end of the second week. Livers and spleens of these animals were pooled and ground in a mortar and inoculated onto Sabouraud dextrose agar. Cultures were kept at room temperature for 6 weeks before being discarded. Sputum from 13 patients were examined and all were found to be negative. O n one patient who had a complement-fixation titre of i : 8 with histoplasmin and I : 6 4 with whole-yeast antigen, 18 examinations of sputum were carried out over a period of 4 months with negative results. The level of titres in the complement-fixation test vary in the same patient at different times. Table I V shows these differences. The relationship between Mantoux, histoplasmin skin test, complementfixation test, and sputum examination for Mycobacterium tuberculosis are shown in Tables V, VI, VII, and V I I I . Twenty-seven patients had a positive complement-fixation test, and" 9 had a positive histoplasmin skin test. I n only one patient was the skin test and sero-

5~

PONNAMPALAM

logical test positive. Eight patients had a positive skin test and negative serological test, and 26 patients had a negative skin test and positive serological test. Considering that a positive histoplasmin skin test and/or a positive complement-fixation test is indicative of infection with Histoplasma capsulatum (Furcolow, I962 ) then 35 patients of a total of i I8 suffering from tuberculosis present evidence of exposure to infection with H. capsulatum--a percentage of 29. 7. Discussion This survey of 227 patients presents an interesting picture and suggests the possibility of histoplasmosis being present in chest hospitals. Forty-five of the 227 patients showed complement-fixation titres of I : 8 or more with either whole-yeast or histoplasmin or both antigens, a percentage of T9"8. Only 24 or io- 5 per cent. showed a positive histoplasmin skin test thereby confirming the findings of other workers in this field (M. L. Furcolow, personal communication, I962), that hyper-sensitivity to intradermal histoplasmin is depressed during illness. As blood for serological tests was collected on the day the skin tests were read the possibility t h a t an elevation of complement-fixation titre occurring as early as 2 days following a histoplasmln skin test in a hypersensitive individual could be ruled out (McDearman and Young, I96o; Nicholas et al., I96I ). T h e precipitin test was carried out on those sera which were anticomplementary. It is unusual for the precipitin test to be positive when the complement-fixation test is negative. All the anticomplementary sera were negative by the precipitin test (Furcolow, personal communication, 1962). Thirteen patients whose sputum was examined for H. capsulatum revealed negative results. Culture of sputum for the fungus is successful in about 2o per cent. of patients with a positive serological titre of I : 32 or less, and in 42 per cent. of patients with a titre of I : 32 or higher (Furcolow, personal communication, I962 ). There is a suggestion in this survey that histoplasmosis m a y co-exist with tuberculosis in some of these patients (Tables V, VI). O f the 227 patients in this series io8 had cavitary disease; a large percentage of patients with a positive serology had evidence of cavitation radiologically. Whether this was due to histoplasmosis or tuberculosis is not certain. Nineteen patients had a positive histoplasmin skin test and negative complement-fixation test. T h e cause for this is uncertain. As an aid in the diagnosis of histoplasmosis both the histoplasmin skin test and the serological test should be carried out because a combination of both these tests is more likely to detect those patients who may be negative if only either of these tests were carried out. While the skin test is positive the serological test should be repeated if negative. What relationship the serological titre bears to different stages of the disease is not certain, neither is it known to what extent cross-agglutination occurs with other fungous antigens. Statistical analysis of the histoplasmin skin tests suggests a uniform distribution of histoplasmin sensitivity among the three major races. ' T h e data were also analysed to determine whether sensitivity varied with age. Since there was no variation between the three race groups, the data for all races were pooled

HISTOPLASMOSIS IN M A L A Y A

55

into 3 groups o f 3 ° years a n d below, 3 1 - 5 o years, a n d those m o r e t h a n 5 ° years. O f the 227 cases e x a m i n e d 6 o u t o f lOi were positive in the 3o a n d below age group, 14 out o f lO2 in the 31-5 ° age group, a n d 4 o u t o f 2 4 in the age g r o u p a b o v e 5 ° years. Statistical analysis o f these d a t a suggest t h a t histoplasmin sensitivity is equally distributed a m o n g the different age groups. Statistical analysis o f the c o m p l e m e n t - f i x a t i o n tests also show no significant v a r i a t i o n a m o n g the different races or b e t w e e n the different age groups.

Summary A survey o f 227 patients f r o m 5 - 6 0 years o f age revealed the presence o f positive histoplasmin skin tests in lO'5 per cent. a n d positive c o m p l e m e n t fixation tests in 19-8 per cent. S p u t u m f r o m 13 o f the 37 patients w h o h a d a positive c o m p l e m e n t - f i x a t i o n test were cultured for H . capsulatum b u t with negative results. Exposure to infection b y the fungus is equally distributed a m o n g the different race a n d age groups. A careful a n d constant w a t c h should be kept for histoplasmosis in all chest hospitals as a certain n u m b e r o f cases m a y be present. I t tends to elude diagnosis unless specially sought as it resembles the clinical picture o f tuberculosis or o t h e r g r a n u l o m a t o u s disease. I t presents a field for f u r t h e r investigation a n d research in M a l a y a . I am indebted to Dr, 1VLL. Fureolow and his staff of the (]omrnunicable Disease (]entre, United States Public Health Service, Kansas Gity, for supplying stock histoplasmin and performing the serological tests. My special thanks to Dr. M. R. J. Snelllng, physician, and 1V[r. H. M. McGladdery, surgeon, Lady Templet Hospital, for allowing me access to their patients; and the sisters and nursing staff for collecting the specimens of sputum. I am also grateful to Professor H. B. Maitland, Head of the Department of Bacteriology, Institute for Medical Research, for reading through the proofs and making the necessary corrections, and Mr. V. A. Ratnam, Assistant Statistician at the Institute, for analysis of data. ADDENDUM

One of the patients with a negative tuberculin and hlstoplasrnln skin tests and a negative sputum for Myeobacterium tuberculosis had a positive complement-fixation test for histoplasmosls and a recent culture of the sputum revealed the presence of Histoplasma capsulatum. REFERENGES ABILOAARD, C. 17.~ TAYLOR, R. e. (1962).Amer. oT. fro#.Med. nyg, If, 5, 667. U.S. PUBL. HEALTH SERVICE (196I). (]o-operativemycoses study, o7. Amer. reed. Ass., i77, 5, 29=. DUNCAN,J. T. (1945). Brit. med. ~7., 2, 715. FURCOLOW,M. L. (I96I , 1962). Personal (]ommunicatlon. MCDEARMAN, S. (]. & YOUNG,I. M. (1960). Amer. J. Clin. Path., 34, 434-38. 1VIASHBURN,J. D., DAWSON,D. F., & YOUNO,J. (I961). Amer. Rev. resp. Dis., 84, 2, 2o8. NICHOLAS, W., WEIR, J. A., KUHN, L. R., (]AMPBBLL, (]. (]., NOLTE, L. G., & HILL, G. B. (1961). Amer. Rev. resp. Dis., 83, =76-79 • PONNAMPALAM,J. T. (1963). Amer. o7. trop. Med. Hyg. In the press. PONNAMPALAM,J. T. (1963). Med. o7. Malaya. In the press.